Diamond Annual Review 2019/20

8 9 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 Macromolecular Crystallography Group Dave Hall, Science Group Leader M acromolecular crystallography (MX) remains at the forefront of understanding the form and function of biologically relevant molecules by revealing their shape and interactions at atomic resolution. The information derived from MX experiments can be complemented by many other life science techniques at Diamond (see in particular the Soft Condensed Matter and Imaging and Microscopy sections of this review) and coupled with experiments in the researcher’s lab to give deeper insight by employing an integrated structural biology approach. At Diamond seven beamlines (I03, I04, I04-1, I23, I24, VMXi and VMXm), alongside the XChem fragment screening facility, the UK XFEL Hub and the Membrane Protein Facility (see the Integrated Facilities and Collaborations section) are dedicated to exploiting the technique of MX for the benefit of the UK structural biology community and researchers fromEurope and further afield. Serial Synchrotron Crystallography (SSX) is available to users now on the Microfocus and Serial MX beamline (I24). In this relatively new area, sample delivery is continuously evolving but fixed targets and the lipidic cubic phase (LCP) extruder are now routine. The first successful light activated experiments for SSX have been carried out using Diamond’s portable laser system, PORTO. Over the course of 2020 further PORTO commissioningwill take place and this, coupled with other techniques, will enable dynamic crystallography at Diamond. The sample requirements for serial experiments can often be daunting; with this in mind some protocols for successful sample preparation have recently been published 1 and we are actively developing approaches that significantly reduce sample consumption by exploiting acoustic drop ejection 2 . Two significant upgrades will take place on I24 in the near future; the current insertion device will be replaced by a cryo-cooled permanent magnet undulator (CPMU), and an Eiger2 detector, equipped with a cadmium telluride (CdTe) sensor, will be installed to complement the existing detector. Biological crystals areradiationsensitive,however,thecombinationoftheCPMUandtheCdTeEiger2 detector will facilitate efficient data collection at higher X-ray energies (more than 20 keV). Operating at higher X-ray energies, in turn, allows significantly more data to be collected frommicrocrystals due to photoelectron escape 3 . The Long-Wavelength MX beamline (I23) has continued to successfully operate in its unique wavelength range. Native phasing, where the intrinsic anomalous signal from, for example, sulphur, for experimental phasing is exploited, is now becoming a routine experiment with an increasing number of successful structure determinations. Access to the potassium absorption edge has provided further insight into the ribosome, one of the fundamental cellular machineries (see I23 highlight).While most ribosome work is now performed by single particle cryo-electron microscopy, this study needed the long wavelength X-rays only available at beamline I23 to unambiguously identify metal ions. Recent updates to the cryogenic sample transfer system and the software user interface, GDA, have increased the robustness and usability of the beamline, which will finally enable users to independently operate the beamline from spring 2020. VMXm, the Versatile MX micro beamline, following first user experiments in late 2018 has been consolidating and undertaking further user experiments. A permanent liquid nitrogen distribution system has been installed in the experimental hutch, which is particularly useful for the sample transfer system. In conjunction with Diamond’s Optics and Metrology Group, microfocus mirror optimisation is ongoing.To date a sub-400 nm vertical X-ray beam at the sample position has been achieved and the functionality of the novel, variable beam size vertical mirror 4 has been confirmed. In parallel, controls and acquisition systems continue to be developed, again to bring functionality and practicality for the final end-users. This work will continue through 2020 to support the transition to a full user programme. Diamond’s I03 and I04 MX beamlines continue to stay at the forefront of detector technologies. The first Eiger2 XE 16M detector was installed on I04 in December 2018. The move from the previously installed detector resulted in a step change for the users and the beamline. Most notably the increase in data acquisition frequency from 25 Hz to 133 Hz initially and then to higher frequencies. I03 installed an identical detector in April 2019. Since early summer 2019, both detectors can operate at 400 Hz in continuous mode and can acquire bursts of more than 28,000 images at 500 Hz. Peak acquisition rates can reach 560 Hz. The significantly smaller pixel size (75 microns versus 172 microns) of the Eiger2 XE 16M in combination with the (almost) zero readout time leads to a noticeable improvement in data quality and allows for better spot separation, throughput has seen several improvements over the last year. The beamline end-station has had its control system upgraded providing greater control and robustness.Datacollectionmethodologiesforfragmentscreeninghave improved with a 30% faster crystal-to-crystal turnaround. Together these upgrades help serve the growing throughput from the XChem lab and an increasingly in- demand user programme from both academia and industry, that is able to offer broader fragment libraries and investigate a larger chemical space for each target of interest. SARS-CoV-2 Structural Work at Diamond Diamond led the way with a specific call for rapid access proposals from the usercommunityforaccesstoitslifesciencefacilitiesinresponsetotheurgentneed to address the COVID-19 outbreak. Additionally, to contribute to the global effort to combat COVID-19, researchers at Diamond in early 2020 solved a structure of the SARS-CoV-2 main protease (M Pro ) at very high resolution (PDB ID 6Y84) and completed an extensive XChem crystallographic fragment screen against it (see I04-1 highlight). The data have been deposited in the Public Protein Database (PDB) and are also available on the dedicated Diamond COVID-19 website 6 . This workwasmade possible by the availability of world classMX instruments and the XChem facility – a unique set of capabilities. The facilities, user programme and training opportunities would not exist without the dedicated beamline scientists, technicians, engineers, software developers, computing support and many other supporting teams at Diamond who make it all happen! 1. Beale J.H. et al. Successful sample preparation for serial crystallography experiments. J. Appl. Cryst . 52 , 1385-1396 (2019). DOI: doi.org/10.1107/S1600576719013517 2. Davy B. et al. Reducing sample consumption for serial crystallography using acoustic drop ejection. J. Sync. Rad. 26 , 1820-1825 (2019). DOI: doi.org/10.1107/S1600577519009329 3. Storm S. L. S. et al. Measuring energy-dependent photoelectron escape in microcrystals. IUCrJ. 7(1) , 129-135 (2020). DOI: 10.1107/S2052252519016178 4. Laundy D. et al. Development of a multi-lane X-ray mirror providing variable beam sizes. Rev. Sci. Instrum. 87(5) , 051802 (2016). DOI: http://dx.doi.org/10.1063/1.4950732 5. Duyvesteyn H. M. E. et al. Glutathione facilities enterovirus assembly by binding at a druggable pocket. Commun. Biol. 3 , 9 (2020). DOI: https://doi.org/10.1038/s42003-019-0722-x 6. https://www.diamond.ac.uk/covid-19/for-scientists.html which is particularly useful when resolving long unit cells. In combination with the SMARGON multi-axis goniometer, operating on both I03 and I04, the Eiger2 XE detector also allows for even faster grid scans.This ability to carry out fast grid scans is used, for example, in automated X-ray centring where the 4M detector regionof interest(ROI) isapplied.Thisallowsfastercollection,processing,analysis and display of results. All processing pipelines have been adapted to deal with the new .h5 file format and new processing hardware has been implemented in order to deal with the heightened computing demand presented by the increased number of pixels. This upgrade has already enabled more efficient use of beamlines I03 and I04, increasing throughput significantly. Moreover, this upgrade also paves the way for new modes of data collection, especially serial approaches. During 2019, unattended data collection protocols and procedures were developed further, resulting in high quality data being collected, fully autonomously, for cryo-cooled samples with no user interaction with the beamlines. Testing with Diamond users has been undertaken on I03 to benefit from their feedback and thus improve upon the systems in place.Tomanage fully autonomous data collection across four beamlines (I03, I04, I04-1 and I24), for thousands of potential users, for hundreds of thousands of samples, including from our XChem lab and from around the world, a robust system is required. Data collection through this system is rapid, in particular when exploiting the new Eiger2 detector properties, as exemplified by the collection this year from virus crystals at very high resolution (>1.7 Å) in around 30s 5 . Unattended data collection will be rolled out to the full user programme through 2020. Staying with the theme of fully automated collection, the room temperature beamline, Versatile MX in situ (VMXi), has continued to mature and develop in conjunction with users from academia and industry. New hardware has been installed, robustness testing undertaken, development of and support for SSX carried out, and pre experiment support for beamtime at the European XFEL provided. The beamline routinely uses its double multilayer monochromator to provide extremely high flux photons. This is exploited by collecting data at extremely high rates using its Eiger2 4M detector. Rotation data sets of 20- 60 o each from crystals in crystallisation plates are taken in 1s. Consequently, the beamline can collect thousands of data sets per day. This is opening up the opportunity to investigate biological structures at near physiological temperatures and enable comparison with data more conventionally collected at cryogenic temperatures.The user programme will develop further through 2020, expanding the opportunities presented by the beamline to a wider community. Beamline I04-1 and the associated XChem facility have continued to offer a greatservicetothegeneraluserprogrammeandthestructure-baseddrugdesign fragment screening programme. A continued focus on robustness and sample